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Methods Mol Biol. 2016;1333:83-100. doi: 10.1007/978-1-4939-2854-5_8.

Analyzing Persister Physiology with Fluorescence-Activated Cell Sorting.

Author information

1
Department of Chemical and Biological Engineering, Princeton University, 205 Hoyt Laboratory, Princeton, NJ, 08544, USA.
2
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
3
Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, USA.
4
Department of Chemical and Biological Engineering, Princeton University, 205 Hoyt Laboratory, Princeton, NJ, 08544, USA. mbrynild@princeton.edu.
5
Department of Molecular Biology, Princeton University, Princeton, NJ, USA. mbrynild@princeton.edu.

Abstract

Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of persister levels in the resulting sorted fractions. These assays provide persister phenotype distributions, which can be compared to the phenotype distributions of the entire population, and can also be used to examine persister heterogeneity. Here, we describe two detailed protocols for analysis of persister physiology with FACS. One protocol assays the metabolic state of persisters using a fluorescent metabolic stain, whereas the other assays the growth state of persisters with use of a fluorescent protein.

KEYWORDS:

Antibiotic; Fluorescence-activated cell sorting (FACS); Persister; Phenotypic heterogeneity; Redox sensor green (RSG); Viable but non-culturable cell (VBNC)

PMID:
26468102
PMCID:
PMC4908830
DOI:
10.1007/978-1-4939-2854-5_8
[Indexed for MEDLINE]
Free PMC Article

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